Middle East respiratory syndrome virus (MERS), a zoonotic lineage C Betacoronavirus discovered in 2012, has caused over 2,000 infections and more than 700 deaths. The emergence of MERS in addition to SARS highlights the public health significance of virulent, emerging coronaviruses (CoVs). MERS is descended from a parental bat CoV (BtCoV), and like other bat borne viruses, is believed to be nonpathogenic in its natural host. The reasons for such disparate outcomes of zoonotic CoV infection between bats and humans represent a gap in knowledge. All CoVs encode lineage specific accessory proteins often with roles in host antagonism of innate responses. MERS accessory proteins NS4a and NS4b are reported to antagonize interferon (IFN)-? induction in overexpression and reporter systems, and we present data herein showing that mutation of either protein confers attenuation of replication to recombinant mutant MERS viruses. However, little is known about the mechanisms of host antagonism during MERS infection, another important gap in knowledge. While NS4a is a dsRNA binding protein that localizes with viral replication/transcription complexes and antagonizes IFN-? mRNA expression, NS4b has no homology with any other protein in NCBI. We used structural modeling to identify MERS NS4b as a LigT-like 2H-phosphoesterases (2H-PE), and like the NS2 protein of lineage A Betacoronavirus MHV, NS4b has 2?,5'-phosphodiesterase (PDE) activity and antagonizes RNase L in the cytoplasm. However, unlike NS2, MERS NS4b has an N-terminal nuclear localization signal (NLS) and localizes primarily to the nucleus. In preliminary data, NS4b also cleaves 3?,5? bonds found in possible RNA substrates, implying other likely nuclear functions. RNA-seq data suggest that NS4b regulates the antiviral host responses as well as programmed cell death pathways, and this may be at least in part by post-transcriptional modification of select mRNAs. We will test the hypothesis that MERS NS4a and NS4b antagonize dsRNA- induced antiviral pathways in the cytoplasm and NS4b is a unique coronavirus protein, which acts enzymatically in the nucleus to down-regulate the abundance of select host mRNAs, further antagonizing antiviral responses. We propose to: 1. Use recombinant MERS mutant viruses to assess NS4a and NS4b-mediated antagonism of dsRNA-induced antiviral pathways in human A549 cells and in primary human airway epithelial cells. 2. Investigate the substrate specificity of NS4b as well as its predicted nuclear role in post-transcriptional regulation of the abundance of select antiviral mRNAs, and explore the possibility that NS4b modulates programmed cell death. 3. Identify bat specific MERS-host interactions by infection of bat derived cell lines and bats in vivo with MERS and NS4a and NS4b mutant viruses. These studies will elucidate the likely multiple functions of the MERS NS4a and NS4b accessory proteins and in the long-term lead to identification of candidate therapeutic targets. In addition, these findings may help explain the highly pathogenic outcome of zoonotic virus infection in humans as compared to their natural hosts.